<i>Zymosan</i>-induced production of TNFα and IL-1α:masking of Siglec-7.

<p>Statistical histogram bar graph showing the percentages of CD14^pos monocyte producing TNF-α (left) and IL-1α (right) either in the absence (gray bars) or in the presence (white bars) of <i>Zymosan</i> and in presence of <i>Zymosan</i> cultured with blocking anti-Siglec-7 Abs (black bars). The intracellular production of TNF-α and IL-1α were evaluated by flow cytometric analysis. Data are representative of 5 independent experiments performed in triplicates (± SD).</p

Intracellular production of pro-inflammatory cytokines and chemokines in monocytes upon engagement of Siglec-7.

<p>(A) Representative flow cytometry dot plot graphs showing the percentage of CD14^pos monocytes, within total PBMCs, producing IL-6, IL-1α, CCL4/MIP-1β, IL-8 and TNF-α after incubation with either the anti-Siglec-7 mAb (lower line) or the matched IgG2b isotype control (upper line). (B) Statistical summary graphs of dot plots with medians (horizontal black bars) and p values showing the percentage of CD14^pos monocytes producing IL-6, IL-1α, MIP-1β, IL-8 and TNF-α in response to either the anti-Siglec-7 mAb (red circles) or the matched IgG2b isotype control (green circles).</p

Binding of pathogens to Siglec-7 Fc chimera.

<p>Representative flow cytometric dot plot graphs showing the binding of goat anti human (GAH) Fc Ab (upper line) and of Siglec-7 Fc chimera (lower line) to <i>Escherichia coli</i> (strains K1 and K12) and <i>Candida albicans</i>. Data are representative of 3 independent experiments.</p

Engagement of Siglec-7 Receptor Induces a Pro-Inflammatory Response Selectively in Monocytes

<div><p>Sialic acid binding immunoglobulin-like lectin-7 (Siglec-7) is a trans-membrane receptor carrying immunoreceptor tyrosine based inhibitory motifs (ITIMs) and delivering inhibitory signals upon ligation with sialylated glycans. This inhibitory function can be also targeted by several pathogens that have evolved to express sialic acids on their surface to escape host immune responses. Here, we demonstrate that cross-linking of Siglec-7 by a specific monoclonal antibody (mAb) induces a remarkably high production of IL-6, IL-1α, CCL4/MIP-1β, IL-8 and TNF-α. Among the three immune cell subsets known to constitutively express Siglec-7, the production of these pro-inflammatory cytokines and chemokines selectively occurs in monocytes and not in Natural Killer or T lymphocytes. This Siglec-7-mediated activating function is associated with the phosphorylation of the extracellular signal-regulated kinase (ERK) pathway. The present study also shows that sialic acid-free <em>Zymosan</em> yeast particles are able to bind Siglec-7 on monocytes and that this interaction mimics the ability of the anti Siglec-7 mAb to induce the production of pro-inflammatory mediators. Indeed, blocking or silencing Siglec-7 in primary monocytes greatly reduced the production of inflammatory cytokines and chemokines in response to <em>Zymosan</em>, thus confirming that Siglec-7 participates in generating a monocyte-mediated inflammatory outcome following pathogen recognition. The presence of an activating form of Siglec-7 in monocytes provides the host with a new and alternative mechanism to encounter pathogens not expressing sialylated glycans.</p> </div

Intracellular production of TNF-α and IL-1α in monocytes upon engagement of Siglec-7 and Siglec-9 and modulation of adhesion molecules in monocytes upon engagement of Siglec-7.

<p>(A) Statistical histogram bar graph showing the percentages of CD14^pos monocyte producing TNF-α (left) and IL-1α (right) in the presence of mAbs cross-linking either Siglec-7 and Siglec-9 or their relative IgG2b and IgG1 isotype controls. Data are representative of 5 independent experiments performed in triplicates (± SD). (B) Representative flow cytometry histogram graphs showing the mean fluorescence intensity (MFI) of ICAM-1 (left) and CD49e (right) on CD14^pos monocyte after incubation with either the anti-Siglec-7 mAb (blue lines) or with the matched IgG2b isotype control (red lines). (C) Statistical summary graphs of box plots with medians and standard deviation showing the MFI of ICAM-1 (left) and CD49e (right) on CD14^pos monocyte after incubation with either the anti-Siglec-7 mAb (blue boxes) or with the matched IgG2b isotype control (red boxes). Data are representative of 5 independent experiments (± SD).</p

Detection of cytokines in PBMC supernatant upon engagement of Siglec-7.

<p>Secretion of IL-6, IL-1α, IL-8, CCL4/MIP-1β, GRO and DAN in the supernatant of PBMCs stimulated with the anti-Siglec-7 mAb (right panel) compared with that of PBMCs incubated with a matched IgG2b isotype control (left panel). The culture medium was collected and analysed using a semi-quantitative protein array detecting simultaneously 507 human soluble proteins. Data shown in this figure are representative of 3 independent experiments.</p

Phosphorylation of ERK upon engagement of Siglec-7 in monocytes.

<p>(A) Representative phospho-flow cytometry dot plot graphs showing the percentage of phosphorylation of p38 MAPK and of ERK 1–2 in freshly purified monocytes incubated with an IgG2b isotype (left column) and anti Siglec-7 mAb (right column) at time 0 (upper line) and after 5 (middle line) and 15 (lower line) minutes of incubation. The number highlighted in bold red within the lower right quadrant of the dot plot graph located in the lower line of right column indicates the phosphorylation of ERK following ligation of Siglec-7. (B) Representative western blot image showing the phoshorylation of ERK 1–2 in freshly purified monocytes stimulated with IgG2b isotype (left) and anti Siglec-7 mAb (right) after 15 minutes of incubation. Data are representative of 3 independent experiments (± SD).</p

Binding of <i>Zymosan</i> to Siglec-7 Fc chimera and to Siglec-7 receptor expressed on freshly putified monocytes.

<p>(A) Representative flow cytometric dot plot graphs showing the binding of goat anti human (GAH) Fc Ab (left), NKp44 Fc chimera (middle) and Siglec-7 Fc chimera (right) to <i>Zymosan</i>. (B) Fluorescent microscopic images of <i>Zymosan</i> particles (gray, left part of the panel) surrounded by PE-labeled Siglec-7 Fc chimera (green, right part of the panel). (C) Fluorescent microscopic images of primary monocytes labeled for Siglec-7 (red) and incubated with <i>Zymosan</i> particles (green) for 5 and 30 minutes. The co-localization is labeled in yellow.</p

Zymosan-induced production of TNFα and IL-1α: silencing of Siglec-7.

<p>(A) Representative graphs showing the intracellular production of TNF-α and IL-1α in monocytes transfected either with non-targeting SiRNA control probes (green line in the histogram graph and dot plot graphs in the upper line of the panel) or with SiRNA duplexes specific for Siglec-7 (red line in the histogram graph and dot plot graphs in the lower line of the panel) in response to <i>Zymosan</i>. MFI: Mean Fluoresence Intensity. Filled gray Histogram: isotype control. (B) Summary graphs of dot plots (left) and statistical summary bar graphs with p values and standard deviation (right) showing the percentages of decrement of TNF-α (left) and IL-1α (right) in monocytes silenced for Siglec-7 compared to monocytes not silenced for Siglec-7 in response to <i>Zymosan</i>. Data are representative of 5 independent experiments (± SD).</p

Correlation between the RET receptor expression and RET transcripts on four different cell lines.

<p>(A) Flow cytometric histogram graphs showing the MFI levels of expression of RET receptor (black line) of 4 different cell lines. Gray shaded histograms represent the isotype controls. (B) Histogram bar graph showing the number of RET mRNA copies produced by the same cell lines displayed in panel A and analyzed within the same time-frame in culture. Values are normalized on SK-N-MC cell line of one experiment and are reported as fold increased in expression (2^−ΔCt) as mean of three independent experiments. Of note, the level of RET receptor expressed on cell membrane significantly correlated with the amount of RET transcripts, as assessed by the Spearman rank test for correlation.</p